Long chain polyunsaturated fatty acids (LC-PUFAs) are PUFAs containing 20 or more than 20 carbon atoms, which can be divided into ω-6 and ω-3 according to the position of the first double bond distance at the C end. ω-3 LC-PUFAs can not be synthesized in human body. It needs to be obtained from diet. It belongs to essential fatty acids, such as EPA and DHA. It is found that ω-3 LC-PUFAs, represented by EPA and DHA, can be used as precursors to synthesize some hormones, with multiple physiological functions and potential medicinal value. So far, the source of ω-3 LC-PUFAs is mainly deep-sea fish, but the LC-PUFAs extracted from it has the disadvantages of poor stability, complex purification process, easy oxidation and so on. In recent years, microorganisms, as a new source of LC-PUFAs, are attracting more and more attention. Marine algae grow through autotrophic, heterotrophic and mixed nutrition and have short growth cycles. It is a primary producer of EPA and DHA and one of the most potential sources of ω-3 LC-PUFAs. Yeast and mold are low in research cost and suitable for mass production. They possess the potential of producing multiple LC-PUFAs. With the rapid development of molecular biology and biotechnology, more and more attention has been paid to the oleaginous fungi in order to produce EPA and DHA through the pathway of fatty acid synthesis in the genetically engineered fungi. However, due to low efficiency and so on, it has not yet been commercialized.
In nature, the synthesis of ω-3 LC-PUFAs is usually initiated by LA and ALA, and finally synthesized into EPA and DHA after a series of enzyme catalysis. Among them, ω-3 fatty acid desaturase is one of the key enzymes in the synthesis of ω-3 LC-PUFAs. It has three histidine rich domains, which can catalyze ω-6 PUFAs to produce corresponding ω-3 PUFAs, such as LA (C18:2), GLA (C18:3), DGLA (C20:3) and ARA (Arachidonic, or written as AA) to ALA (C18:3), SDA (C18:4), ETA (C20:4) and EPA (C20:5).
It is found that ω-3 fatty acid desaturase from different sources has different catalytic efficiency for fatty acids with different carbon chain lengths. At present, known ω-3 fatty acid desaturase derived from algae and plants can only catalyze 18C ω-6 PUFAs such as LA and GLA. The ω-3 fatty acid desaturase FAT 1 derived from the Caenorhabditis elegans can simultaneously use 18C and 20C PUFAs as substrates, but the catalytic activity of 20C PUFAs substrates is very low. Subsequently, Pereira (et al.) found that ω-3 fatty acid desaturase sdd17 from Saprolegnia diclina has no catalytic activity for 18C PUFAs, but can catalyze 20C ARA to EPA, and the conversion rate is 25.9%. Similarly, ω-3 fatty acid desaturase OPIN 17 from Phytophthora infestans can not use 18C PUFAs as substrate, but the conversion rate of ARA is 30.94%.
In recent years, Xue (et al.) have isolated three ω-3 fatty acid desaturase, PaD 17 from Pythium aphanidermatum, PsD 17 from Phytophthora sojae and PrD 17 from Phytophthora ranmorum, and have heterologous expression in recombinant yeast (Identification and characterization of new Δ-17 fatty acid desaturases, Appl Microbiol Biotechnol (2013) 97:1973-1985). These three enzymes not only catalyze 18C PUFAs but also catalyze 20C PUFAs, and prefer to catalyze 20C substrate ARA. Through further study of these fatty acid desaturase, it is found that they have high conversion rate to ARA at normal temperature, so the biological accumulation of EPA at normal temperature is realized. This kind of fatty acid desaturase preference uses 20C ω-6 LC-PUFAs as substrate to catalyze the synthesis of ω-3 LC-PUFAs, so they can catalyze ARA to produce EPA directly and efficiently, which is of great significance for the production of ω-3 LC-PUFAs by biosynthetic method. In order to improve the production efficiency, it is particularly important to screen the fatty acid desaturase which can efficiently synthesize EPA.
On the other hand, Mortierella alpina is an oleaginous fungus with a lipid accumulation that can reach 50% of the stem weight of the cell. It has been applied to the industrial production of Arachidonic acid (AA, C20:4), and its edible oil has been evaluated by the safety of FDA. In addition to synthesizing AA, Mortierella alpina also has certain ability to synthesize Eicosapentanoic acid (EPA, C20:5). EPA belongs to ω-3 long chain polyunsaturated fatty acids (LC-PUFAs), which plays an important role, such as promoting the development of the mammalian brain and the formation and repair of nerve tissue, and preventing asthma, cancer, depression, obesity, immune disorder, and cardiovascular disease. But these fatty acids can not be synthesized by human body, and they need to be absorbed from foods rich in ω-3 LC-PUFAs (such as deep-sea fish oil). Owing to overfishing and environmental pollution, the ω-3 LC-PUFAs provided by deep-sea fish is unable to meet the demand of the market, the production of ω-3 LC-PUFAs by microorganism has become a research hotspot.
However, obtaining an oleaginous fungus with higher EPA yield, especially Mortierella alpina, remains the expectation of technicians in this field.